Stewart A K, Sutherland D R, Nanji S, Zhao Y, Lutzko C, Nayar R, Peck B, Ruedy C, McGarrity G, Tisdale J, Dubé I D
Department of Medicine, The Toronto Hospital, Ontario, Canada.
Hum Gene Ther. 1999 Aug 10;10(12):1953-64. doi: 10.1089/10430349950017310.
We conducted a phase I hematopoietic stem cell (HSC) gene-marking trial in patients undergoing autologous blood or marrow stem cell transplant for the treatment of multiple myeloma. Between 500 and 1000 ml of bone marrow was harvested from each of 14 myeloma patients and 1 syngeneic donor. A mean of 3.3x10(9) cells per patient were plated in 20 to 50 long-term marrow culture (LTMC) flasks and maintained for 3 weeks. LTMCs were exposed on days 8 and 15 to clinical-grade neo(r)-containing retrovirus supernatant (G1Na). A mean of 8.23x10(8) day-21 LTMC cells containing 5.2x10(4) gene-marked granulocyte-macrophage progenitor cells (CFU-GM) were infused along with an unmanipulated peripheral blood stem cell graft into each patient after myeloablative therapy. Proviral DNA was detected in 71% of 68 tested blood and bone marrow samples and 150 of 2936 (5.1%) CFU-GM derived from patient bone marrow samples after transplant. The proportion of proviral DNA-positive CFU-GM declined from a mean of 9.8% at 3 months to a mean of 2.3% at 24 months postinfusion. Southern blots of 26 marrow and blood samples were negative. Semiquantitative PCR analysis indicated that gene transfer was achieved in 0.01-1% of total bone marrow and blood mononuclear cells (MNCs). Proviral DNA was also observed in EBV-transformed B lymphocytes, in CD34+ -enriched bone marrow cells, and in CFUs derived from the latter progenitors. Gene-modified cells were detected by PCR in peripheral blood and bone marrow for 24 months after infusion of LTMC cells. Sensitivity and specificity of the PCR assays were independently validated in four laboratories. Our data confirm that HSCs may be successfully transduced in stromal based culture systems. The major obstacle to therapeutic application of this approach remains the overall low level of genetically modified cells among the total hematopoietic cell pool in vivo.
我们对接受自体血液或骨髓干细胞移植治疗多发性骨髓瘤的患者进行了一项I期造血干细胞（HSC）基因标记试验。从14例骨髓瘤患者和1例同基因供体中分别采集500至1000毫升骨髓。每位患者平均3.3×10⁹个细胞接种于20至50个长期骨髓培养（LTMC）瓶中，并培养3周。在第8天和第15天，将LTMC暴露于临床级含neo(r)逆转录病毒上清液（G1Na）中。在清髓治疗后，将平均8.23×10⁸个第21天的LTMC细胞（其中含有5.2×10⁴个基因标记的粒-巨噬细胞祖细胞（CFU-GM））与未经处理的外周血干细胞移植物一起输注到每位患者体内。在68份检测的血液和骨髓样本中，71%检测到前病毒DNA，移植后从患者骨髓样本中获得的2936个CFU-GM中有150个（5.1%）检测到前病毒DNA。输注后3个月时，前病毒DNA阳性CFU-GM的比例平均为9.8%，到24个月时平均降至2.3%。对26份骨髓和血液样本进行的Southern印迹分析均为阴性。半定量PCR分析表明，基因转移在总骨髓和血液单个核细胞（MNC）的0.01%至1%中实现。在前病毒DNA转化的B淋巴细胞、富集CD34⁺的骨髓细胞以及源自后者祖细胞的CFU中也观察到前病毒DNA。输注LTMC细胞后24个月，通过PCR在外周血和骨髓中检测到基因修饰细胞。PCR检测的灵敏度和特异性在四个实验室中得到独立验证。我们的数据证实，HSC在基于基质的培养系统中可成功转导。该方法治疗应用的主要障碍仍然是体内整个造血细胞池中基因修饰细胞的总体水平较低。
